FOOD PRODUCT WHICH HAS BEEN FORTIFIED IN IRON CONTENT,

Abstract

A food product which has been fortified in iron content, having an iron content of at least 5 ppm and comprising iron-containing nanoparticles, wherein the nanoparticles are stabilised by means of a biopolymer provides good bioavailability and stability.

Full Text

F7841/V
FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
IRON FORTIFIED FOOD PRODUCT AND ADDITIVE
HINDUSTAN UNILEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed

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IRON FORTIFIED FOOD PRODUCT AND ADDITIVE
FIELD OF THE INVENTION
The invention generally relates to the field of fortified food products. More in particular, it relates to the fortification of a food product with iron. The invention also relates to an additive for the fortification and supplementation of food and other products with iron and to a method of preparing the same.

BACKGROUND TO THE INVENTION
Iron is an essential trace element in animal and human nutrition. It is a component of heme in hemoglobin and of myoglobin, cytochromes and several enzymes. The main role of
iron is its participation in the transport, storage and utilization of oxygen.
Iron deficiency was and remains a common nutritional problem not only in the developing world but also in the
industrialized countries. Inadequate intake of dietary iron causes the high incidence of anemia which nutritional surveys have identified among children, adolescents and women. Since the human body does not produce minerals, it is totally dependent on an external supply of iron, either nutritional or
supplementary. The importance of adequate iron intake is recognized during the whole life of the human being. The recommended daily allowance for iron intake is from 10 to 18 mg per day, and is dependent on age and sex. Children, women up to the time of menopause, and expectant and nursing mothers
are in the group with higher requirements of iron.
In general, water-soluble inorganic minerals are likely to impair the stability of the food product, and it is hard to

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Another problem in iron fortification is the capacity of iron to promote destructive free-radical reactions, which can result in off- flavors. Thus, the addition of soluble iron sources to fat containing products - mostly products with a high level of unsaturated fatty acids - cause flavor changes due to lipid oxidation. Iron promoted oxidation not only affects the organoleptic properties of foods and beverages, but also undesirably affects the nutritional quality of these products. These interactions can be also enhanced during heat treatment, such as pasteurization or sterilization.
Finally, from a technical point of view, soluble iron salts can also cause corrosion of processing equipment.

As alternatives to the soluble sources of iron, which are highly bioavailable but lead to an undesirable flavor and/or color, insoluble iron sources such as elemental iron, ferric pyrophosphate, etc. may be used. These forms of iron cause
little or no discoloration and off-flavor problems but are poorly bioavailable. In addition, added to drinks and liquid beverages they may cause sedimentation, which could make the mineral not available to the consumer because it remains in the package, or lost of transparency, if added to clear
products.
Also, the efficiency of the uptake of iron from food fortified with large particles of water-insoluble iron salts or elemental iron, the bioavailability and bioacessability, remain a problem due to slow dissolution of the mineral.

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Finally, from a technical point of view, the use of insoluble
large particles of insoluble iron salts can cause abrasion and
severe damage of the mixing and processing equipment.
So far, few attempts have been made to simultaneously address these very complex issues. For example, EP-B-870 435 (Taiyo Kagaku) discloses a mineral-containing composition having improved dispersion stability and comprising enzymatically decomposed lecithin and water-insoluble mineral, preferably
Fe(III) or ferric pyrophosphate. The use of enzymatically decomposed lecithin is essential for achieving the desired dispersion stability. A major drawback of these compositions is that the emulsifier lecithin has to be present. It is known that lecithin has not very pleasant taste. In addition, the
use of emulsifier makes the product particularly costly and not appealing to the consumer.
Products containing lecithin are "Generally Recognised As Safe" (GRAS) under 21 CFRa 184.1400 and specifications of the
Food Chemicals Codex. Lecithin products that have been
modified sometimes require special labelling. For example, when enzymatically modified, the phrase "Enzymatically Modified Lecithin" should appear on labelling. Finally, lecithin is known to vary significantly in quality from batch
to batch causing extra difficulties in food processing.
In many cases the unnecessary use of emulsifiers is not desirable. Therefore, it is desirable to develop a nutritional additive that fulfils the above-mentioned requirements for stability without the necessity of using enzymatically-decomposed lecithin.

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It is therefore an object of the present invention to provide an iron fortified food product and iron-containing additive, which overcomes one or more of the above mentioned drawbacks. Surprisingly, it has now been found that this object can be achieved by the food product according to the invention, having and iron content of at least 5 ppm (R. F. Hurrell, Preventing Iron Deficiency Through Food, Fortification, Nutrition Reviews, Vol. 55, No. 6), comprising iron-containing nanoparticles stabilised with biopolymers.
SUMMARY OF THE INVENTION
According to a first aspect, the invention provides a food product which has been fortified in iron, having iron content of at least 5 ppm, comprising iron-containing nanoparticles, wherein the nanoparticles are stabilised by means of a biopolymer.
According to a second aspect, there is provided an iron containing additive for use in the food and other products according to the invention, in the form of an iron-containing nanoparticles having a diameter of 5 - 1000 nanometer, wherein the nanoparticles are stabilised by means of a biopolymer.
According to a third aspect, there is provided a process for preparing the iron-containing additive of the invention and according to a fourth aspect, there is provided a process for making the food product of the invention.

DETAILED DESCRIPTION OF THE INVENTION
The invention regards an iron-fortified food product having an iron content of at least 5 ppm iron (Fe). The food product

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comprises iron in the form of iron-containing biopolymer-
stabilised nanoparticles. Nanoparticles are defined for the
purpose of this invention as particles stabilised by the
presence of protective biopolymer. They have a particle size
of about 5 to 1000 nanometer. The compositions of the
invention contain biopolymer-stabilised iron containing
nanoparticles, which have an effective average particle size
of less than about 1000 nm. In a preferred embodiment of the
invention, the biopolymer stabilised iron containing
nanoparticles have an effective average particle size of less than about 900 nm, preferably less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm,
less than about 100 nm, less than about 75 nm, or even less than about 50 nm.
The effective average particle size can be measured using techniques that are well known in the art, such as electron microscopy or light scattering techniques. The nanoparticles may be crystalline, polycrystalline or amorphous.
The iron containing nanoparticles used in the present invention are stabilised by means of a biopolymer and their
derivatives, such as, polyamides (e.g. proteins and poly(amino acids)), polysaccharides (e.g. cellulose, starch and xanthan), organic polyoxoesters synthesized by bacteria and eukaryotic organisms (e.g. poly(hydroxyalkanoic acids), poly(malic acid), polylactides, polyglycolide, polyanhydrides, polyesteramides
and cutin), polythioesters, polyphosphate, polyisoprenoids (e.g. natural rubber or Gutta Percha), polyphenols (e.g. lignin or humic acids), and nucleic acids such as ribonucleic acids and deoxyribonucleic acids. The most preferred

carboxymethylcellulose (CMC-Na), propylene glycol alginate ester (PGA) and beet-derived pectin (BD-pectin), OSA starch. These may be used alone or in combination.
Incidentally, the biopolymer can be used together with other nonionic or negatively charged surfactants. It is desired that the surfactant is usually used so as to be contained in the mineral additive of the present invention in the range of from 0 to 20% by weight.

The amount of the biopolymer to be used may be generally about 0.01 to 10 wt.%, preferably 0.1 to 5 wt.%, and preferably around 1% wt. with respect to the total amount of non-dried product containing nanoparticles, but these ranges do not
restrict the scope of the invention because they may vary depending on differences in the type of biopolymer and concentration of nanoparticles. The weight ratio of biopolymer to iron-containing nanoparticles is generally at least about 1:10,000 or higher (e.g. more biopolymer in comparison to
nanoparticle mass).
The advantages of using the biopolymer-stabilised iron-containing nanoparticles according to the present invention are the excellent chemical stability in respect to interaction
with other elements, oxidation, complexion activity, and
colour change due to the low concentration of free iron ions these than soluble iron salts. Very importantly, due to the presence of stabilising biopolymer, these particles are compatible with many products containing other biopolymers.

Furthermore, due to their low chemical activity, these iron containing nanoparticles allow multiple fortification with

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vitamins, other minerals such as Ca, Zn, Mn, Mg, Cu, Se and other micro-nutrients.
Due to their very small particle size, sedimentation is very slow or completely negligible in comparison to large
particles, which provides excellent physical stability of liquid and semi-liquid products.
In addition, the nanoparticles have excellent dispersibility in aqueous phases, including emulsions and gels, and in products comprising the same.
Due to their small particle size, the mineral compositions have a good bioavailability and bioacessability in comparison to large particles of the same compound.
Due to their small size and low solubility, these substances don not cause adverse organoleptic effects, such bad (metallic) taste, chalkiness and sandiness.
Furthermore, due to their small size, these substances do not have significant abrasion effect on the equipment.
The iron-fortified food products of the present invention can be advantageously in the form of beverages, (dry) soups, fat spreads, (yoghurt or protein) drinks, dressings or cereal products like bread.
A second aspect of the invention is an iron-containing additive for use in the food or other products as iron
supplement according to any one of the preceding claims, in the form of iron containing nanoparticles of iron insoluble inorganic or organic salt, or mixtures thereof, and having a

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particle size of 5 to 1000 nanometer, wherein the nanoparticles are stabilised by means of a biopolymer.
The iron-containing additive preferably comprises a low-
soluble salt having a Ks of 10"7 or less. By low soluble we
mean a Ks, where Ks is the solubility product, of 10-7 or
less.
The forms of the water-insoluble minerals generally include inorganic salts, organic salts, and the like. The inorganic salts include, for example, ferrous and ferric oxides (e.g. Fe2O3, Fe3O4), hydroxides (e.g. FeOOH, Fe(OH)3), carbonates (e.g. FeCO3, Fe2(CO3)3, phosphates ( e.g. Fe3(PO4)2, FePO4, Fe2P2O7, Fe4(P2O7)3, FeNH4PO4) or other inorganic insoluble iron 15 salts or mixtures thereof. Examples of organic low-soluble salts are iron - partially hydrolysed proteins, iron phytate or other sufficiently low soluble organic salts of iron. Each of those inorganic salts can be used alone or in admixture of two or more salts.
More preferably, the low-soluble iron salt is selected from the group of iron phosphates, more preferably pyrophosphate and iron orthophosphate or mixtures thereof.
The iron containing food product is prepared by mixing the iron-containing additive as dispersed in liquid or dried form using a suitable mixing process known in the art.
The amount of iron in the food product is at least 5 ppm iron (Fe), but preferably it is at least 10, 20, 50 or even 100 ppm.

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According to another embodiment, the iron-containing additive is prepared by chemical homogeneous or inhomogeneous precipitation in the presence of the biopolymer or a mixture of biopolymers. The precipitation can be achieved by fast mixing, using any suitable fast mixing process, of two
solutions or (liquid-in-liquid, liquid-in-gas, gas-in-liquid, or solid-in-liquid or mixtures) dispersions containing iron ions and counter ions that form insoluble iron salt, respectively. The biopolymer can be present in either or in both phases. The pH of the final product can be from 2 to 8, preferably between 6 and 7. Preferably, the biopolymer is present in the system containing ions that do not interact strongly with the biopolymer.
The resulting iron-containing biopolymer-stabilised
nanoparticles can be separated from the mother liquid and dried e.g. using spray or freeze drying. Or the can be concentrated or directly dried together with the side products. Preferably, the side products should be Na, K or
ammonium salts.
The resulting iron-containing biopolymer-stabilised nanoparticles can be crystalline, polycrystalline or amorphous. In the preferred embodiment, the biopolymer- stabilised nanoparticles are amorphous or polycrystalline.
Finally, the additive according to the invention, comprising iron-containing nanoparticles could be further utilized in a wide variety of fields such as cosmetics, animal feed additives, plant fertilizers, pharmaceutical products, personal and home care products.

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The animal feeds containing the iron-containing nanoparticles
of the present invention include, for example, feeds for pets,
domestic animals, cultured fishes, and the like.
Cosmetics containing the iron-containing nanoparticles of the present invention include lotion; milky lotion; bathing agents; detergents such as cleansing agents; dentifrices, skin creams and the like.
Industrial products containing the iron-containing
nanoparticles of the present invention include iron-based catalysts, agricultural purposes, for slugs and snails control, sheet materials for walls or floors, additive to polymers and resins.

The invention will mow be further illustrated by means of the following, non-limiting examples.
Example 1
Whey protein stabilized iron(II) pyrophosphate nanoparticles
A solution containing 0.01M pyrophosphate and 1% wt. whey protein isolate was prepared by dissolving sodium pyrophosphate decahydrate and whey protein isolate (trade name: BiPro 95, manufactured by Danisco Food International) in demineralized water. An iron (II) solution containing 0.02M Fe was prepared by dissolution of ferrous chloride tetrahydrate in demineralized water.
The iron (II) solution was then quickly added the pyrophosphate-whey protein solution prepared above with vigorous stirring. The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation of iron (II) pyrophosphate

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nanoparticles - a greenish suspension that does not sediment for several hours was formed. The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying. The electron microscopy analyses revealed particle sizes of less than 1000 nm.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.
To prepare Product I, the whey protein-stabilized ferrous pyrophosphate nanoparticles formed in the solid phase were collected and re-suspended in ion-exchanged water to give concentrated ferrous pyrophosphate slurry.
To prepare Product II, the entire reaction mixture was dried.
Example 2
Whey protein stabilized iron(III) pyrophosphate nanoparticles
A solution containing 0.015M pyrophosphate and 1% wt whey protein isolate was prepared by dissolving sodium
pyrophosphate decahydrate and whey protein isolate (trade name: BiPro 95, manufactured by Danisco Food International) in demineralized water. An iron (III) solution containing 0.02M Fe was prepared by dissolution of ferric chloride hexahydrate in demineralized water.
The iron (III) solution was then quickly added the pyrophsophate-whey protein solution prepared above with vigorous stirring. The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation white suspension of iron (III) pyrophosphate nanoparticles that does not sediment for several

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hours was formed. The electron microscopy analyses revealed particle sizes of less than 1,000 nm.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.
To prepare Product III, the whey protein-stabilized ferrous pyrophosphate nanoparticles formed in the solid phase were collected and then re-suspended in ion-exchanged water to give a concentrated ferric pyrophosphate slurry.
To prepare Product IV, the entire reaction mixture was dried.
Example 3
Gum Arabic stabilized iron(II) pyrophosphate nanoparticles
A solution containing 0.075M pyrophosphate and 0.5% wt gum Arabic was prepared by dissolving sodium pyrophosphate decahydrate and Gum Arabic (manufactured by Sigma -Aldrich) in demineralized water. An iron (II) solution containing 0.015M Fe was prepared by dissolution of ferrous sulfate heptahydrate in demineralized water.
The iron (II) solution was then quickly added the pyrophosphate-Gum Arabic solution prepared above with vigorous
stirring. The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation of a greenish suspension of iron (II) pyrophosphate nanoparticles that does not sediment for several hours. The electron microscopy analyses revealed particle
sizes of less than 1,000 nm.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.

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To prepare Product V, the Gum Arabic-stabilized ferrous pyrophosphate nanoparticles formed in the solid phase were collected and the resulting complex was then re-suspended in ion-exchanged water to give a concentrated ferrous pyrophosphate slurry.
To prepare Product VI, the entire reaction mixture was dried.
Example 4
Gum Arabic stabilized iron(III) pyrophosphate nanoparticles
A solution containing 0.075M pyrophosphate and 0.5% wt gum Arabic was prepared by dissolving sodium pyrophosphate decahydrate and Gum Arabic (manufactured by Sigma -Aldrich) in demineralized water. An iron (III) solution containing 0.01M Fe was prepared by dissolution of ferric chloride hexahydrate in demineralized water.
The iron (III) solution was then quickly added the pyrophosphate-Gum Arabic solution prepared above with vigorous stirring. The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation of white suspension of iron (III) pyrophosphate nanoparticles that does not sediment for several hours was formed. The electron microscopy analyses revealed particle sizes of less than 1,000 nm.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.
To prepare Product VII, the Gum Arabic-stabilized ferrous pyrophosphate nanoparticles formed in the solid phase were collected, and the resulting complex was then re-suspended in

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ion-exchanged water to give a concentrated ferrous pyrophosphate slurry.
To prepare Product VII, the entire reaction mixture was dried.
Comparative example 1
A solution containing 0.01M pyrophosphate was prepared by dissolving sodium pyrophosphate decahydrate in demineralized water. An iron (II) solution containing 0.02M Fe was prepared by dissolution of ferrous chloride tetrahydrate in demineralized water.
The iron (II) solution was then quickly added the pyrophosphate solution prepared above with vigorous stirring.
The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation of greenish iron (II) pyrophosphate precipitate, which immediately sediments. The electron microscopy observation revealed the formation of large irregular
aggregates.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.
To prepare Reference Product A, the ferrous pyrophosphate precipitate formed in the solid phase was collected and re-suspended in ion-exchanged water, to give a concentrated pyrophosphate slurry.

water. An iron (III) solution containing 0.02M Fe was prepared by dissolution of ferric chloride hexahydrate in demineralized water.
The iron (III) solution was then quickly added the
pyrophosphate solution prepared above with vigorous stirring. The pH of the resulting mixture was not further adjusted. The reaction self-terminated after several minutes after the formation of white-yellow iron (III) pyrophosphate precipitate, which immediately sediments. The electron microscopy observation revealed the formation of large irregular aggregates.
The resulting reaction mixture was subjected to solid-liquid separation by centrifugation, to concentration, or to drying.
To prepare Reference Product B, the ferric pyrophosphate precipitate formed in the solid phase was collected and re-suspended in ion-exchanged water, to give concentrated ferric pyrophosphate slurry.

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CLAIMS
1. Food product which has been fortified in iron content, having an iron content of at least 5 ppm, comprising iron containing nanoparticles, wherein the nanoparticles are stabilised by means of a biopolymer.
2. Food product according to claim 1, wherein the nanoparticles comprise a low-soluble iron salt having a Ks of 10"7 or less.
3. Food product according to claim 2, wherein said low-soluble iron salts is an inorganic salt selected from the group consisting of ferrous and ferric oxides (e.g. Fe2O3, Fe3O4) , hydroxides (e.g. FeOOH, Fe(OH)3), carbonates (e.g. FeCO3, Fe2(CO3)3), phosphates (e.g. Fe3(PO4)2, FePO4, Fe2P207, Fe4(P2O7)3/ FeNH4PO4) or other inorganic insoluble iron salts or mixtures thereof.
4. Food product according to claim 2, wherein said low-soluble iron salts is selected from the group consisting of organic low-soluble salts of iron with partially hydrolysed proteins, phytate, organic acids, or other sufficiently low soluble organic salts of iron.
5. Food product according to claim 1-4, comprising a mixture of inorganic and organic salts.
6. Food product according to any one claims 1-5, wherein the biopolymer is selected from the group of polyamides, polysaccharides, organic polyoxoesters synthesized by bacteria and eukaryotic organisms, poly(malic acid), polylactides, polyglycolide, polyanhydrides, polyesteramides and cutin,

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polythioesters, polyphosphate, polyisoprenoids, polyphenols, and nucleic acids.
7. Food product according to any one claims 1-5, wherein the biopolymer is a protein, a peptide, a poly-amino acid, a polysaccharide or mixtures thereof.
8. Food product according to claim 7, wherein the biopolymer is a protein.
9. Food product according to claim 8, wherein the protein is selected from the group consisting of soy protein, whey protein and casein.
10. Food product according to claim 7, wherein the biopolymer is a polysaccharide, preferably a gum.
11. Food product according to any one of the preceding claims, the form of fat spread, a (protein) drink, an instant protein drink, a beverage or a dressing.
12. Iron-containing additive for use in the food or other products according to any one of the preceding claims, in the form of iron containing nanoparticles having a particle size of 5 to 1,000 nanometer, wherein the nanoparticles are stabilised by means of a biopolymer.
13. Iron-containing additive according to claim 12, wherein the nanoparticles comprise a low-soluble iron salt having a Ks of 10"7 or less.
14. Iron-containing additive according to claim 13, wherein said low-soluble iron salt is selected from the group

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consisting of low-soluble iron inorganic salts selected from the group of ferrous and ferric oxides (e.g. Fe2O3, Fe3O4) , hydroxides (e.g. FeOOH, Fe(OH)3), carbonates (e.g. FeCO3, Fe2(CO3)3), phosphates ( e.g. Fe3(P04)2, FePO4, Fe2P2O7, Fe4(P2O7)3, FeNH4PO4) or other inorganic insoluble iron salts or mixtures thereof.
15. Iron-containing additive according to claim 13, wherein said iron salt is selected from the group consisting of low-soluble iron inorganic salts of organic such low-soluble salts of iron with partially hydrolysed proteins, phytate, organic acids, or other sufficiently low soluble organic salts of iron.
16. Iron-containing additive according to claim 12-15, comprising a mixture of inorganic and organic salts.
17. Iron-containing additive according to claim 12-16, wherein the biopolymer is selected from the group consisting of polyamides, polysaccharides, organic polyoxoesters synthesized by bacteria and eukaryotic organisms, poly(malic acid), polylactides, polyglycolide, polyanhydrides, polyesteramides and cutin, polythioesters, polyphosphate, polyisoprenoids, polyphenols and nucleic acids.
18. Iron-containing additive according to claim 17, wherein the biopolymer is a protein.
19. Iron-containing additive according to claim 18, wherein the protein is selected from the group consisting of soy protein, whey protein and casein.
20. Iron-containing additive according to claim 12, wherein the biopolymer is a polysaccharide.

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21. Iron-containing additive according to claim 12, wherein said polysachcaride is selected from the group consisting of locust bean gum, tamarind seed polysaccharide, gellan gum, xanthan gum, guar gum, tara gum, gum arabic, kalaya gum, carrageenan, agar soybean polysaccharides and mixtures thereof.
22. Iron-containing additive according to claim 12, further comprising an auxiliary non-polysaccharide stabilizer selected from the group consisting of glycol alginate esters, methoxy pectin (HM-pectin), sodium carboxymethylcellulose (CMC-Na), propylene glycol alginate ester (PGA), and beet-derived pectin (BD-pectin), starch, OSA starch or combinations thereof.
23. Process for preparing the food product according to any one of claims 1-11, comprising the steps of adding or mixing the iron-containing additive in solid or dispersed form with the food products.
24. Iron-containing additive according to claim 12-13, wherein the low-soluble iron salt is obtained by a method for forming salts by homogeneous, heterogeneous or mixed precipitation and in the presence of a biopolymer.